Nephron number and new imaging techniques for histology specimens

Norbert Gretz

Medical research center

Klinikum Mannheim

University Heidelberg

MRI experience

Combining new tools to assess renal function and morphology: a holisticapproach to study the effects of aging and a congenital nephron deficit.

Geraci S, Chacon-Caldera J, Cullen-McEwen L, Schad LR, Sticht C, Puelles VG, Bertram JF, Gretz N.

Am J Physiol Renal Physiol. 2017 Sep 1;313(3):F576-F584.

Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging.

Heilmann M, Neudecker S, Wolf I, Gubhaju L, Sticht C, Schock-Kusch D, KrizW, Bertram JF, Schad LR, Gretz N.

Nephrol Dial Transplant. 2012 Jan;27(1):100-7.

3

3D imagingafter

optical tissue clearing (OTC)

and/orexpansion

Silvestri et al. J. Biomed. Opt. (2016). doi:10.1117/1.JBO.21.8.081205

Optical tissue clearing methods

Principal: removal of lipids + refractive index adjustment(how fast light propagates through material)

OTC methods

StrategyMethods Time to clear

Morphologicalalterations

Immunstainingdemonstrated

Solvent based• BABB• 3DISCO• iDISCO

• Days• Hours-days• Hours-days

• Shrinkage• Shrinkage• Shrinkage

• Yes• Limited• Yes

Acqueus solvent• CLEAR T• SeeDB• TDE

• Hours-days• Days• Days-weeks

• No• No• No

• Yes• No• Yes

Hyperhydration• Scale/A• Scale/S• CUBIC

• Weeks• Days• Days

• Expansion• No• Expansion

• Yes• Yes• Yes

Gel embedding• CLARITY• PACT• PARS

• Days• Days-weeks• Days

• Slight expansion• Slight expansion• No

• Yes• Yes• Yes

PBS 3DISCO uDISCO SeeDB FRUIT SCALE/S CUBIC PACT

Adapated from: Xu et al., J Biophotonics (2019)

Recent publications from our groupA Novel Optical Tissue Clearing Protocol for Mouse Skeletal Muscle toVisualize Endplates in Their Tissue Context.

Williams MPI, Rigon M, Straka T, Hörner SJ, Thiel M, Gretz N, Hafner M, Reischl M, Rudolf R.

Front Cell Neurosci. 2019;13:49

• MYOCLEAR

A cationic near infrared fluorescent agent and ethyl-cinnamate tissueclearing protocol for vascular staining and imaging.

Huang J, Brenna C, Khan AUM, Daniele C, Rudolf R, Heuveline V, Gretz N.

Sci Rep. 2019;9:521

A cationic near infrared fluorescent agent and ethyl-cinnamatetissue clearing protocol for whole body vascular imaging. J. Huang, C. Brenna, A. ul Maula Khan, C. Daniele, R. Rudolf, N. Gretz, Scientific Reports2019

Optical Tissue Clearing (OTC) Methods for Modern Histology

workflow

optical tissue

clearing

http://zmf.umm.uni-heidelberg.de/restricted/

scanning: confocal, light sheet (2x SP8)

• Microscopy (1 mouse kidney)• confocal special objectives (60 h)

• light sheet (23 min)

• Data volume• (voxel size) (1 mouse kidney)

• 200 nm 83 TB

• 1 µm 756 GB

• 10 µm 770 MB

14

2 SP8microscopes

Scientific datastorage (SDS)

High performancecluster (HPC)

bwVISU- Linux- Docker - Container- Deep learning- LAS X- Lightning

heiCLOUD- LAS X- Lightning

Researcher - remote

Light wires

• Mouse Kidney - 1mm slice;

• Removal of ECi by ethanol and rehydration after ECiclearing;

• Antibody staining (1st: Antibody against Nephrin; 2nd: Alexa Fluo 647) ;

• Imaging by Confocal Microscope with 20x and 88% glycerol as mounting solution

05.12.2018 Cinzia Brenna 16

3D ANTIBODY STAINING AFTER ECi TISSUE CLEARING

05.12.2018 Cinzia Brenna 17

C.Brenna – Images acquired by Confocal Microscope

Kidney

0

100

200

300

400

500

600

700

Histologicalslide (IHC)

Denaturated(Non-ExM)

Expanded(ExM)

Glo

me

rula

r Si

ze (

µm

)

Glomerular Size (max diameter)

Podocin - IHC

200µm

550*

*#

ExM – Kidney - Expansion Factor

100 µm

n=13Frozen section

Expansion factor

1,8

4,3

10 cm

500µm

denaturated

expanded

Gelatedkidney

3 cm

02.03.2019 Yalcin Kuzay 19

NephrinStaining

See Original file: Z:\Yalcin Kuzay\Expansion Microscopy\Eosin-Hematoxylin-Methyl Green- Perfused Rat\228-Hematoxylin-PFA

There are also 3D images

Control Rat-1Podocin20x Water immersion

2

20 x

Zoom-in from glomeruli to podocyte foot processes

Cai, R. et al. (Ertürk group)Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull–meninges connections. Nat. Neurosci. 22, 317–327 (2019).

3D imaging of a mouse as a whole

24

3D imagingafter

optical tissue clearing (OTC)

and/orexpansion